Project Summary/Abstract Synovial sarcoma is a soft-tissue tumor that typically affects children and young adults under thirty years of age. Metastatic synovial sarcoma carries a particularly poor prognosis and there is currently no targeted therapy for this cancer. Synovial sarcoma is driven by a fusion oncoprotein, SS18-SSX (also called SYT-SSX), which fuses the majority of the SS18/SYT protein to a short, seventy-eight amino acid tail derived from SSX. Although the association of SS18-SSX with synovial sarcoma has been established for decades, the precise molecular function of this fusion was unknown until several years ago. In 2013, our collaborator Dr. Cigall Kadoch published a groundbreaking study that revealed that the normal SS18 protein is a component of the mammalian SWI/SNF chromatin remodeler complex (BRG1/BRM Associated Factors (BAF) complex), which plays a crucial role in regulating gene expression. However, in synovial sarcoma, the SS18-SSX fusion evicts normal SS18 and the BAF47 subunit from the BAF complex causing their degradation. SS18-SSX, through as yet undefined mechanisms (which will be defined in RP1), hijacks the BAF complex and re-targets it to sites in chromatin normally repressed by the Polycomb Repressive Complex 2 (PRC2). SS18-SSX initiates the transcriptional activation of these BAF neo-target genes, many of which are pro-proliferative genes such as SOX2, and in doing so, drives an oncogenic state. Synovial sarcomas are dependent on the SS18-SSX fusion for sustained proliferation, however, overexpression of wildtype SS18 is sufficient to block synovial sarcoma cell proliferation. This is consistent with our biochemical data demonstrating that SS18 and SS18-SSX function strictly as monomers that inhabit mutually exclusive BAF complexes. As such, these two molecular species directly compete for incorporation into the BAF complex to elicit opposing downstream cellular effects (oncogenic vs. tumor suppressive). The molecular balance between these two proteins and how it can be leveraged to develop targeted therapies is the subject of this research proposal. In Aim 1 we will use a multidisciplinary approach combining genetics, biochemistry and next-generation sequencing technologies to characterize the precise molecular mechanisms as to how wildtype SS18 blocks synovial sarcoma proliferation, by defining crucial functional domains, transcriptional targets and the SS18 gene-dosage required to arrest sarcoma growth. In Aim 2 we will identify targets and develop small molecules aimed at stabilizing wildtype SS18 in synovial sarcoma cells. These studies will follow on the Shilatifard?s lab success in identifying a new therapeutic paradigm for MLL- fusion childhood leukemias. We will develop fluorescent SS18 protein stability reporters and use them to screen genome wide CRISPR Cas9 libraries to identify factors involved in SS18 degradation. Collectively these studies will provide insight into the dysregulated chromatin state of synovial sarcoma and enable the development of screening strategies for novel therapeutics that will have relevance to other fusion onco-protein driven cancers.